ISSN 1313 - 8820 Volume 5, Number 4 December 2013

2013

Editor-in-Chief Tsanko Yablanski Faculty of Agriculture Trakia University, Stara Zagora Bulgaria Co-Editor-in-Chief Radoslav Slavov Faculty of Agriculture Trakia University, Stara Zagora Bulgaria Editors and Sections Genetics and Breeding Atanas Atanasov (Bulgaria) Ihsan Soysal (Turkey) Max Rothschild (USA) Stoicho Metodiev (Bulgaria) Nutrition and Physiology Nikolai Todorov (Bulgaria) Peter Surai (UK) Zervas Georgios (Greece) Ivan Varlyakov (Bulgaria) Production Systems Dimitar Pavlov (Bulgaria) Dimitar Panaiotov (Bulgaria) Banko Banev (Bulgaria) Georgy Zhelyazkov (Bulgaria) Agriculture and Environment Georgi Petkov (Bulgaria) Ramesh Kanwar (USA) Product Quality and Safety Marin Kabakchiev (Bulgaria) Stefan Denev (Bulgaria) Vasil Atanasov (Bulgaria) English Editor Yanka Ivanova (Bulgaria)

Scope and policy of the journal Agricultural Science and Technology /AST/ – an International Scientific Journal of Agricultural and Technology Sciences is published in English in one volume of 4 issues per year, as a printed journal and in electronic form. The policy of the journal is to publish original papers, reviews and short communications covering the aspects of agriculture related with life sciences and modern technologies. It will offer opportunities to address the global needs relating to food and environment, health, exploit the technology to provide innovative products and sustainable development. Papers will be considered in aspects of both fundamental and applied science in the areas of Genetics and Breeding, Nutrition and Physiology, Production Systems, Agriculture and Environment and Product Quality and Safety. Other categories closely related to the above topics could be considered by the editors. The detailed information of the journal is available at the website. Proceedings of scientific meetings and conference reports will be considered for special issues. Submission of Manuscripts All manuscripts written in English should be submitted as MS-Word file attachments via e-mail to [email protected]. Manuscripts must be prepared strictly in accordance with the detailed instructions for authors at the website http://www.uni-sz.bg/ascitech/index.html and the instructions on the last page of the journal. For each manuscript the signatures of all authors are needed confirming their consent to publish it and to nominate on author for correspondence. They have to be presented by a submission letter signed by all authors. The form of the submission letter is available upon from request from the Technical Assistance or could be downloaded from the website of the journal. Manuscripts submitted to this journal are considered if they have submitted only to it, they have not been published already, nor are they under consideration for publication in press elsewhere. All manuscripts are subject to editorial review and the editors reserve the right to improve style and return the paper

for rewriting to the authors, if necessary. The editorial board reserves rights to reject manuscripts based on priorities and space availability in the journal. The articles appearing in this journal are indexed and abstracted in: EBSCO Publishing, Inc. and AGRIS (FAO). The journal is accepted to be indexed with the support of a project № BG051PO0013.3.05-0001 “Science and business” financed by Operational Programme “Human Resources Development” of EU. The title has been suggested to be included in SCOPUS (Elsevier) and Electronic Journals Submission Form (Thomson Reuters). Internet Access This journal is included in the Trakia University Journals online Service which can be found at www.uni-sz.bg. Address of Editorial office: Agricultural Science and Technology Faculty of Agriculture, Trakia University Student's campus, 6000 Stara Zagora Bulgaria Telephone.: +359 42 699330 +359 42 699446 http://www.uni-sz.bg/ascitech/ Technical Assistance: Nely Tsvetanova Telephone.: +359 42 699446 E-mail: [email protected]

Volume 5, Number 4 December 2013

ISSN 1313 - 8820

2013

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 5, No 4, pp 394 - 399, 2013

Nutrition and Physiology

Impaired pancreatic function in mulard ducks with experimental aflatoxicosis I. Valchev1*, N. Grozeva2, D. Kanakov1, Ts. Hristov1, L. Lazarov1, R. Binev1, Y. Nikolov1 1

Department of Internal Non-Infectious Diseases, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria Department of General and Clinical Pathology, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria

2

Abstract. Changes in blood concentrations of some biochemical indices (alpha amylase, lipase, insulin and blood glucose) as well as in pancreas morphology were monitored in mulard ducks with experimental aflatoxicosis В1. The experiments were conducted with 4 groups of 20 10-day-old mulard ducks: group I – control, fed a standard compound feed according to the species and the age; group II – experimental, whose feed was supplemented with 0.5 mg/kg AFB1, group III – experimental, supplemented with 0.8 mg/kg AFB1 and group IV – experimental, supplemented with 0.5 mg/kg AFB1 and 2 g/kg Mycotox NG. The duration of the experiment was 42 days. By the 21st day of the experiment, ducks from groups II and III showed reduced activity of enzymes and blood glucose. The observed changes tended to become more pronounced by the 42nd day of the trial. Histopathologically, the pancreas exhibited intralobular swelling, disorganisation and disintegration of glandular acini, various extents of dystrophic changes, mononuclear infiltrates as well as necrobiotic changes depending on the dose of ingested toxin. The supplementation of contaminated feed with a mycosorbent (Mycotox NG) reduced statistically significantly the changes in blood biochemical parameters as well as the severity and frequency of observed histological lesions.

Keywords: Аflatoxin В1, pancreas, mallard ducks.

Introduction Aflatoxins are secondary toxic metabolites produced by Aspergillus flavus, A. parasiticus and A. nomius (Miller, 1995; Kim et al., 2001; Huwing et al., 2001). These fungi are natural contaminants of poultry feeds (Edds and Bortell, 1983). The global distribution of aflatoxins causes significant economic losses to poultry industry and livestock husbandry (Miller, 1995). They are frequently detected as contaminants of cereal crops (wheat, corn, soy, sorghum), which are among the commonest ingredients of poultry feeds. Aflatoxins are encountered as field cereal contaminants before the harvest and then, during storage and after technological processing and packaging (Council for Agricultural Science and Technology, 1989). The five main varieties encountered in cereal crops and animal foodstuffs are aflatoxin B1, B2, G1, G2 and M1. AFB1 is the most biologically active compound among all aflatoxins (Busby and Wogan, 1981; Kubena et al., 1990; Barjesteh et al., 2010). The detrimental effects of aflatoxins in poultry are characterised with reduced growth rate, lower feed conversion, immunosuppressive effects, changes in relative weights of viscera, changes in haematological and blood biochemical parameters (Kubena et al., 1993a,b, 1998; Oguz et al., 2000; Rosa et al., 2001; Basmacioglu et al., 2005; Sakhare et al., 2007; Soliman et al., 2008). Aflatoxins induce morphological alterations in vital organs such as the liver and the kidneys. Liver morphological changes are characterised with increase relative weight, paler colour (yellowish), as well as dystrophic changes in hepatocytes and hyperplasia of bile ductules (Dafala et al., 1987; Ledoux et al., 1999; Ortatatli and Oguz, 2001). In renal parenchyma, aflatoxins induce hyperaemia, haemorrhages, dystrophic and necrotic changes (Sakhare et al., 2007; Soliman et al., 2008). They inhibit the activity of enzymes involved in the metabolism of carbohydrates, proteins, lipids and nucleic acids in * e-mail: [email protected]

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birds, as well as blood total protein, cholesterol and urea concentrations (Dewegowda and Murthy, 2005). Maurice et al. (1983) demonstrated that oral intake of low concentrations (50 or 100 µg/kg b.w.) aflatoxins with feeds could disturb the normal metabolism in broiler chickens. The production of aflatoxins in animal feeds increased in ambient temperatures higher than 27°С, humidity > 62% and feed moisture > 14% (Russo and Yanong, 2002). Birds exposed to the toxic effect of aflatoxins are prone to parasitic, bacterial and viral diseases (Pier, 1981). The detoxication of aflatoxin-contaminated feeds is a major problem faced by poultry industry (Leeson et al., 1995). The currently used methods for detoxication of feeds are chemical, physical and biological, as well as involve the use of mycosorbents (Council for Agricultural Science and Technology, 1989; Abo-Norag et al., 1995; Parlat et al., 1999). Unfortunately, most of these approaches are expensive, time-consuming and only partly efficient. Natural and synthetic zeolites (Kececi et al., 1998; Oguz et al., 2000), bentonites (Ibrahim et al., 2000; Rosa et al., 2001), Mycosorb (Basmacioglu et al., 2005), and other adsorbents are preferred due to their ability to bind aflatoxins in the gastrointestinal tract and thus, to reduce their absorption. The main advantages of adsorbents are their low cost, easy application through supplementation to feeds and reliable effect. According to the American Food and Drug Agency (FDA) permissible dietary aflatoxin concentrations for poultry are up to 20 ppb (Aravind et al., 2003). Although the intake of feeds contaminated with aflatoxins in most cases leads to liver damage, the pancreatic function could be also affected through damage of pancreatic acinar cells. After experimentally induced aflatoxicosis in quails (Chang and Hamilton, 1982) and chickens (Bailey et al., 1998; Kubena et al., 1993a,b, 1998; McKenzie et al., 1998; Ledoux et al., 1999) a higher relative weight of the pancreas, haemorrhages and congestive events were

established in quails (Jakhar and Sadana, 2004) and degenerative alterations – in ducks (Agag, 2004). Contamination of animal feeds and foods for human consumption with aflatoxins is an issue of particular significance posing a serious threat to economy and animal and human health. The effects of these mycotoxins on the metabolism of carbohydrates, proteins and fat result in inevitable changes in the histostructure of the pancreas and concentrations of pancreatic enzymes. The purpose of the present study was to follow out the changes in some biochemical parameters specific for pancreatic functions (α-amylase, lipase, glucose and insulin) as well as the changes occurring in the morphology of the gland.

Material and methods The experiment was performed with eighty 10-day-old female mulard ducks. They were divided into 4 groups with 20 birds in each. The experimental design was as followed: · Group І – control. Mulards from the control group were fed balanced compound feed according to their age, manufactured at the Zoohraninvest, Stara Zagora. They were fed pelleted starter, grower and finisher feeds. · Group ІІ – experimental. Mulards received the standard feed supplemented with 0.5 mg/kg aflatoxin В1. · Group III – experimental. Mulards received the standard feed supplemented with 0.8 mg/kg aflatoxin В1. · Group IV – experimental. Mulards received the standard feed supplemented with 0.5 mg/kg aflatoxin В1 and 2 g/kg Mycotox NG (Ceva Sante Animale, France). Aflatoxin В1 used in this experiment was produced by Aspergillus flavus (99% purity) and purchased from Sigma-Aldrich, Germany. In experimental groups, the feed was ground before being mixed with aflatoxin for better homogenisation. The mulards were reared under optimal microclimatic conditions, equal for all groups. In the beginning of the experiment, ambient air temperature was 35°C and decreased by 1°C daily until the 15th day to attain 20°C by the 28th day; thereafter it was kept at +18°C, with relative air humidity 60–75% (Ordinance, 44/2006). The duration of the light day was 24 h throughout the trial. The control and experimental groups were housed in separate 4 m2 sections in the same premise. The sections were bedded with a 5 cm-layer of clean dry wood shavings. During the first week, the feeding width was 1 сm and thereafter – 10 cm. Blood samples were collected from v. metatarsalis medialis by the 21st and 42nd day of the trial in sterile heparinised vacutainers (FL

medical, Italy) for analysis of alpha amylase, lipase, blood glucose and insulin concentrations. Blood was centrifuges within 30 min from collection at 1500×g for 10 min at 4 oC. Plasma was separated, immediately frozen and kept at –20°С until analysed. Alpha amylase, lipase and blood glucose were analysed on an automated biochemical analyser BS–120, Mindray, China. Insulin levels were assayed using the electroluminescence method (ECLIA) on an automated biochemical analyser Elecsys 2010, Roche. After the end of the experiment, the control and treated birds were euthanised by cervical dislocation. Pancreas specimens for histology were fixed in 10% solution of neutral formalin. Samples were embedded in paraffin after dehydration in ethanol series. The paraffin blocks (5 m) were cut on a microtome Leica RM 2235 and stained with haematoxylin-eosin. The experiment was approved by the Ethics Commission and Animal Welfare at theTrakia University, Stara Zagora (Permit 42.10.10.2011). Data were statistically processed by one-way analysis of variance (ANOVA), and the level of statistical significance was determined by the Tukey-Kramer test (p0.05).

Results Blood biochemical analyses Table 1 presents the changes in studied blood biochemical indices (α-amylase, lipase, insulin and glucose) in control ducks and those treated with AFB1 either independently or in combination with the tested mycosorbent. The concentrations of pancreatic enzymes (α-amylase and lipase) were statistically significantly lower (p